1. Field of the Invention
The invention relates generally to methods, apparatus and compositions, useful for detecting the presence of caffeine in a liquid sample, and more specifically, for detecting the presence of caffeine in a beverage.
2. Description of the Related Art
Competitive ligand-receptor assays. Ligand-receptor assays take advantage of the ability of bioreagents to identify and quantify minute amounts of a wide range of substances, also referred to as analytes, with a high degree of specificity and sensitivity. Competitive ligand-receptor assays are one variant of ligand-receptor assays in general. In competitive ligand-receptor assays, analyte substances in the sample compete with another substance, for example a signal-producing substance, for a limited number of binding sites on the counterpart of the ligand-receptor pair. After the binding has taken place, the amount of other substance bound to the counterpart is detected by any of several means. The signal intensity of competitive ligand-receptor assay is in an inverse relationship with the concentration of analyte present; thus, a sample with no analyte will give a maximum signal intensity, and a sample with a range of analyte concentration will produce less than a maximum signal. Thus, conventional competitive ligand-receptor assays, acting alone, have a maximum sensitivity in a narrow analyte concentration range and require external calibration and standardization.
An additional disadvantage of traditional competitive ligand-receptor assays is that they require external calibration. This disadvantage manifests itself in so-called semi-quantitative assays, where a “yes” or “no” is indicated by the assay based on the presence or absence of a predetermined concentration of analyte. Such semi-quantitative competitive ligand-receptor assays are difficult to perform without external calibration, thus limiting their usefulness in a variety of important market segments. Due to the inverse relationship between signal intensity and analyte concentration, all but the most concentrated samples will give a signal in the assay, and therefore a standard curve (or at least one control point with a known standard) must be run in parallel with the sample assay to interpret accurately any reading of the sample assay. For example, an optical density reading of 0.5 in a competitive immunoassay using enzymes as a signal producing system is meaningless. However, if the user runs a known standard of, for example, 10 micrograms per milliliter of analyte and obtains a reading of 1.0, then the sample with the reading of 0.5 can be said to be more concentrated than the 10 microgram per milliliter sample. The need for standardization has severely limited the practical usefulness of current competitive ligand-receptor assays by requiring several runs of the assay to determine one sample concentration. One of the major disadvantages of the requirement for outside calibration is the concomitant reduction of precision and accuracy of each assay due to inter-assay variability in the calibration process. Furthermore, while there are commercially-available immunochromatographic test strip versions of the ligand-receptor competitive assay available that do not require external calibration, these assays are designed to give a positive indication for the analyte at the least sensitive portion of the analyte concentration versus signal intensity curve. Thus, these immunochromatographic test strips are to be interpreted as positive for analyte in the sample when no signal is seen at the test line. As one skilled in the art would recognize, the precision of the determination of analyte concentration is compromised in such an assay.
Immunoassays. One flavor of ligand-receptor assay is an immunoassay. Various known formats exist for immunoassays, including immunochromatographic test strips for detecting small molecule analytes. One format uses a competitive immunoassay, for which the result is revealed as two lines (negative result) or one line (positive result). Another format displays a single line as an indication of a positive result. Drawbacks of these formats include a very low dose-response ratio at the positive/negative cutoff concentration for some analytes, multiplicity of necessary reagents, high cost of production, and uncertain adaptability to the concentration range of interest for some analytes.
Caffeine detection assays. Immunoassays can be used to detect various analytes, including assays using anti-caffeine antibodies to detect the presence of caffeine. Existing clinical laboratory analyses and test strip formats using traditional immunoassay techniques for caffeine provide varying results. In the laboratory setting, the scientific literature includes methods such as electrometric determination in which a caffeine-specific electrode is prepared from a caffeine-picrylsulfonate ion-pair complex dissolved in octanol; fluorimetric determination in which a buffered solution of caffeine is oxidized with N-bromosuccinimide and then reacted with dimethyl o-phenylenediamine followed by a fluorescence measurement at 480 nm; colormetric determination in which an ethenolic solution of caffeine is oxidized by potassium bromate, dried and then redissolved in dimethylformamide followed by an absorbance measurement at 500 nm; Fourier Transform Infrared Spectrophotometry (FTIR); thin-layer/gas chromatography; enzyme-linked immunosorbent assays (ELISA) in which a caffeine-containing sample of plasma or serum is dissolved in a buffered solution and incubated in a vessel where it competes with peroxidase-labeled caffeine for the binding sites on caffeine antibodies followed by detection of a visible color change with the addition of o-phenylenediamine; immunoassay of theophylline with cross-sensitivity for caffeine; and immunoliposome assay of theophylline with cross-sensitivity for caffeine.
There are a number of commercially available lateral-flow type tests disclosing methods for the detection of large or small analytes, using either “typical” competitive inhibition to produce negative or indirect reporting results, i.e., reduction of signal with increasing analyte concentration, or producing positive or direct reporting results, i.e., increase in signal with increasing analyte concentration. For example, U.S. Pat. Nos. 5,229,073; 5,591,645; 4,168,146; 4,366,241; 4,855,240; 4,861,711; 5,120,643; 4,703,017; 5,451,504; 5,451,507; 5,798,273; 6,001,658; and 6,699,722. However, these types of commercially available lateral-flow type tests use either “typical” competitive inhibition to produce negative or indirect reporting results, or produce positive or direct reporting results, which share the drawbacks described above. Available tests also may take too long to produce a result to be viable.
It is relatively easy to determine the presence of a wide variety of compounds using analytical chemistry techniques. However, such methods often are not available, or practical, for individual consumers seeking to determine the presence or absence of certain compounds in their food and beverages. For example, although “decaffeinated” coffees, teas, and soft drinks have become increasingly popular, the average consumer has no way of verifying the absence (or presence) of caffeine in such beverages when receiving them in restaurants and other public and private settings.
The rise in consumption of decaffeinated beverages has resulted in part from the health concerns of ingesting excessive amounts of caffeine. Caffeine is a bitter crystalline alkaloid. There are a variety of biological effects and symptoms caused by the ingestion of caffeine including tachycardia, diuresis, headaches, decrease in fine motor coordination, insomnia, and central neurological stimulation. Excessive amounts of caffeine can make people tense, irritable, and, in some cases, elevate the heart rate to unsafe levels. Caffeine can also irritate the alimentary canal. It is common for people diagnosed with sensitive stomachs and colons, as well as other medical conditions, to be required to refrain from ingesting caffeine as part of their medical treatment. Pregnant women may not drink any caffeinated beverages for fear of a teratogenic effect. Both men and women avoid caffeinated beverages because caffeine is a known diuretic. Also, as people age, they become increasingly sensitive to the effects of caffeine. However, an individual requesting a decaffeinated beverage can not be fully certain of the reduced level or absence of caffeine in the beverage.
The present invention addresses these and other deficiencies of the prior art as described more fully below.